Various diseases of the nervous system involving myelin damage are known. Some, such as, for example, encephalomyelitis, can be produced in typical laboratory test mammals by sensitizing with certain fractions of material from mammalian central nervous tissue, such as bovine spinal cord. Such a disease is experimental allergic encephalomyelitis. An example of an encephalitogen is the large protein, about 142 amino acids, which is described in an article by Eylar and Hashim, in Vol. 61, Proceedings of the National Academy of Science, August 1968, pgs. 644-650. This protein was referred to by the author as the "A1" protein. In the article there is described enzymatic digestion of the A1 protein to produce shorter chain fragments, and the authors found that two such fragments, which they labelled "E" and "E1", of 16 and 26 amino acids respectively contained all of the encephalitogenic activity of the whole A1 protein.
Protection against diseases often involves introducing the protective agents into the animal before challenge, and generally this results in the formation of antibodies which then prevent the disease or ameliorate symptoms. In autoimmune demyelinating diseases, the role played by antibodies has not been conclusively determined. It appears that a number of other factors, not well known, are probably involved.
The present invention involves a process for separating the crude basic protein obtained from mammalian central nervous system tissue, such as bovine spinal cord tissue, into fractions of different encephalitogenic properties by chromatography.
The crude basic protein utilized in this invention is isolated from mammalian central nervous system tissue after the tissue is defatted by extraction with an organic solvent or solvent mixture capable of dissolving lipids in the presence of water, examples are ketonic solvents, chloroform-methanol, fluorocarbon-ethanol, etc. It should be noted that dehydration usually accompanies the defatting process; however, this is not an essential step. The actual isolation of the crude basic protein involves a simple acid extraction of the defatted mammalian central nervous system tissue. Generally, the tissue is successively extracted with water, aqueous NaCl and water at low pH's. Dilute acid of a pH between 1.5-2.5 extracts the crude basic protein which is then salted out, dissolved in water, dialyzed and freeze-dried. Other standard acid extraction methods may be employed with the provision that the conditions not be such as to cause any significant degree of degradation of the tissue. It should be further noted that the central nervous system tissue for these extractions can be spinal cord tissue or brain tissue and can be obtained from any number of different mammalian species including but not limited to horse, mouse, guinea pig, rabbit, monkey and man. Aside from the practical limitation of availability the only requirement is that the donor animal must be of sufficient age so that development in the central nervous system has progressed to the stage of myelination, since the crude basic proteins are subfractions of myelin.
The invention more specifically relates to the separation of fractions by chromatographing the crude basic protein and the rechromatographing of sub-fractions or reaction products of sub-fractions obtained from central nervous system tissue. The terms used to designate the various fractions isolated are meant only to show the relation of the components to each other.
The crude basic protein is dissolved in about 0.005M to about 0.015M sodium carbonate in about 0.01M to about 0.09M ammonium hydroxide solution and carefully placed on the column containing the sodium form of carboxymethylcellulose. The column is next eluted with about 0.01M to about 0.03M sodium chloride in about 0.01M to about 0.09M ammonium hydroxide followed by a developing solution in which the concentration of the sodium chloride is increased to about 0.1M to about 0.5M. Two fractions are isolated after desalting and freeze drying, S1, a material having a weight average molecular weight of 2,000 to 4,000 and low encephalitogenic activity, and S2 a material having a weight average molecular weight of 8,000 to 10,000 and a high degree of encephalitogenic activity. It has also been found that treatment of the S2 fraction with about 5N to 7N strong acid, preferably hydrochloric acid also gives rise to S1 material. The acid concentration is not critical since a lowering of concentration of the acid may be compensated for by increasing the time of treatment. The S2 fraction, after acid treatment followed by removal of the acid, usually by use of gel filtration of an ion exchange resin, and drying, preferably freeze-drying, was chromatographed in the same manner as the crude basic protein. In fact, crude basic protein itself can be treated directly with strong acid to form increased amounts of S1 material which can be isolated as described. The S1 material isolated from the material treated with acid has a weight average molecular weight of 2,000 to 4,000 and is of low encephalitogenic activity. While it very closely resembles the S1 material obtained directly from the crude basic protein, the two materials probably are not identical although very closely related. A second fraction S3, also obtained from the acid treatment of S2 is of about the same weight average molecular weight as S2, that is 8,000 to 10,000, but of a slightly lesser encephalitogenic activity. This S3 material can be treated again with strong acid and processed as described to yield additional S1 material.
It has also been found that the S1 fractions can be rechromatographed on the ammonium form of carboxymethylcellulose. The S1 fraction, dissolved in about 0.005M to about 0.02M ammonium chloride in about 0.005M to about 0.02M ammonium hydroxide, is applied to the column which is then eluted with the same ammonium chloride-ammonium hydroxide solution. After a period of time the concentration of the eluent is increased to about 0.1M to about 0.5M ammonium chloride-ammonium hydroxide solution. The fractions are desalted and freeze dried.
The S1 fraction obtained directly from the crude basic protein of central nervous system tissue gives three sub-fractions S1A1 the first to be eluted from the column, S1A2, the second, and S1A3, the third. These fractions have weight average molecular weights of 2,000 to 4,000 and are of low encephalitogenic activity.
The S1 fraction obtained from a fractionation of the acid treated S2 fraction gives four fractions (see FIG. 1). These components are eluted in the following order, S1A1, S1A2 (which appears to be a mixture of S1A2a, S1A2b) and S1A3. The weight average molecular weight of these components is 2,000 to 4,000 and these materials possess weak encephalitogenic activity. The components obtained from the rechromatographing of the S1 obtained from the acid treated S2 material and the S1 obtained directly from the crude basic protein of central nervous system tissue are very similar in all their characteristics; however, some minor structural differences probably are present.
Another aspect of the invention is a rather remarkable protection which results when large doses of the low molecular weight materials are given to animals, such as guinea pigs, sensitized with an encephalitogen, for example 15 .mu.g of S2 material. Untreated guinea pigs sensitized in this way typically develop encephalomyelitic symptoms over a period of two to four weeks, whereas animals treated with massive doses, 1-mg. doses repeated ten times from the 7th to the 18th day for a total of 10 mg., are protected, in some cases completely and in other cases to a marked extent. The mechanism whereby the treatment with low encephalitogenic material in massive doses after challenge with sufficient material to cause serious disease and/or death is not known. It is, therefore, not intended to limit this aspect of the present invention to any theory of why such great protection results from treatment with weakly encephalitogenic material after sufficient sensitization to normally result in a serious disease onset. The results, however, are quite dramatic, and of course very useful, because this permits effective treatment of useful mammals after sensitization which would otherwise cause serious disease or death. When it is attempted to treat with smaller doses before challenge there is also sometimes some amelioration but not the dramatic protection obtainable with the doses after challenge.
Others have observed and reported before such protection using highly encephalitogenic proteins, but achieving such protection with weakly encephalitogenic materials is a novel and important part of the present invention.
Because of the convenience and cheapness of using guinea pigs and their high sensitivity they are very useful as laboratory test animals. Thus specific data in the experiments is shown with this animal instead of with some of the larger, more useful mammals, such as horses, dogs and the like.
Because of the ease of producing experimental encephalomyelitis in quinea pigs, this is the form of demyelinating disease which is specifically and quantitatively described. It should be understood, however, that the invention is not limited thereto and is applicable also to the whole spectrum of diseases which result in degradation or inflamation of the myelin sheaths of nerves.